Test media and quantitative or qualitative method for identification and differentiation of biological materials in a test sample

ABSTRACT

A test medium and method for detecting, quantifying, identifying and differentiating up to four (4) separate biological materials in a test sample. A test medium is disclosed which allows quantifying and differentiating under ambient light aggregates of biological entities producing specific enzymes, which might include general coliforms,  E. coli,  Aeromonas, and Salmonella or Shigella in a single test medium. A new class of nonchromogenic substrate is disclosed which produce a substantially black, non-diffusible precipitate. This precipitate is not subject to interference from other chromogenic substrates present in the test medium. In a preferred form, the substrates are selected such that  E. coli  colonies present in the test medium show as substantially black, general coliforms colonies show in the test medium as a blue-violet color, Aeromonas colonies present in the test medium show as a generally red-pink color, and Salmonella or Shigella colonies show as a generally teal-green color. Other microorganisms and color possibilities for detection and quantification thereof are also disclosed. An inhibitor and method for making a test medium incorporating the inhibitor are disclosed.

BACKGROUND OF THE INVENTION

The present invention relates to a test medium and method for thedetection, quantification, identification and/or differentiation ofbiological materials in a sample which may contain a plurality ofdifferent biological materials.

Bacteria are the causative factor in many diseases of humans, higheranimals and plants, and are commonly transmitted by carriers such aswater, beverages, food and other organisms. The testing of thesepotential carriers of bacteria is of critical importance and generallyrelies on “indicator organisms.” Borrego et al., Microbiol. Sem.13:413-426, (1998). For example, Escherichia coli (E. coli) is a gramnegative member of the family Enterobacteriaceae which is part of thenormal intestinal flora of warm blooded animals, and its presenceindicates fecal contamination (e.g., raw sewage). Even though moststrains of E. coli are not the actual cause of disease, their presenceis a strong indication of the possible presence of pathogens associatedwith intestinal disease, such as cholera, dysentery, and hepatitis,among others. Consequently, E. coli has become a prime indicatororganism for fecal contamination, and as a result, any method whichdifferentiates and identifies E. coli from other bacteria is veryuseful.

Others members of the family Enterobacteriaceae, commonly referred to as“general coliforms,” especially the genera Citrobacter, Enterobacter andKlebsiella, are also considered to be significant indicator organismsfor the quality of water, beverages and foods. Therefore, tests toidentify and differentiate general coliforms from E. coli are also veryuseful. Also, various species of the genus Aeromonas have been shown tonot only be potential pathogens, but to have a correlation to otherindicator organisms (Pettibone et al., J. App. Microbiol. 85:723-730(1998)). Current test methods to identify, separate and enumerateAeromonas spp. from the very similar Enterobacteriaceae have beenlacking and most of the current methods utilizing enzyme substrates donot separate Aeromonas spp. from Enterobacteriaceae due to their almostidentical biochemical profiles. Any method that depends upon theidentification of general coliforms by means of a β-galactosidasesubstrate either does not differentiate Aeromonas spp. from generalcoliforms or eliminates Aeromonas from the sample by the use of specificinhibitors (antibiotic such as cefsulodin). Brenner et al., Appl. Envir.Microbio. 59:3534-44 (1993). They do not differentiate, identify andenumerate Aeromonas along with E. coli and general coliforms. Landre etal., Letters Appl. Microbiol. 26:352-354(1998). Improved test methods toeffectively identify, separate and enumerate such bacterial types areneeded, and there is a continuing search for faster, more accurate,easier to use and more versatile test methods and apparatus in thisarea.

Numerous test methods have been utilized to determine, identify andenumerate one or more indicator organisms. Some of these test methodsonly indicate the presence or absence of the microorganism, while othersalso attempt to quantify one or more of the particular organisms in thetest sample. For example, a qualitative test referred to as thePresence/Absence (or P/A) test, may be utilized to determine thepresence or absence of coliforms and E. coli in a test sample. A testmedium including the β-galactosidase substrateO-nitrophenyl-β-D-galactopyranoside (ONPG), and the β-glucuronidasesubstrate 4-methyl-umbrelliferyl-β-D-glucuronide (MUG), is inoculatedwith the test sample. To differentiate the general coliforms from E.coli, this test relies on the fact that generally all coliforms produceβ-galactosidase, whereas only E. coli also produces β-glucuronidase inaddition to β-galactosidase. If any coliforms are present (including E.coli), the broth medium turns a yellow color due to the activity of thegalactosidase enzyme on the ONPG material, causing the release of adiffusible yellow pigment. If E. coli is present, the broth medium willdemonstrate a blue fluorescence when irradiated with ultraviolet rays,due to the breakdown of the MUG reagent with the release of thefluorogenic dye caused by the production of the glucuronidase enzyme.These reactions are very specific, and allow the presence of bothcoliforms in general, as well as E. coli to be identified in a singlesample. A disadvantage of this test is that it is not directlyquantitative for either bacterial type, since both reagents producediffusible pigments. A second disadvantage is that there may a falsepositive coliform reaction if Aeromonas spp. are present in the testsample. This has been shown to be possible even when there areinhibitors present to supposedly prevent this from occurring (Landre etal., Letters Appl. Microbiol. 26:352-354 (1998)). The test also requiresspecific equipment for producing the ultraviolet rays. Further, thistest may only be used to detect coliforms and E. coli. Other importantmicroorganisms, such as the strain E. coli 0157 which is glucuronidasenegative, are not detected, nor are othernon-galactosidase-glucuronidase producing microorganisms.

The Violet Red Bile Agar (VRBA) method has been used to determine thequantity of both coliform and E. coli in a test sample. The test mediumused in this method includes bile salts (to inhibit non-coliforms),lactose and the pH indicator neutral red. As coliforms (including E.coli) grow in the medium, the lactose is fermented with acid production,and the neutral red in the area of the bacterial colony becomes a brickred color. The results of this test are not always easy to interpret,and in order to determine the presence of E coli, confirming follow-uptests, such as brilliant green lactose broth fermentation, growth in ECbroth at 44.5° C. and streaking on Eosin Methylene Blue Agar (EMBA),must be performed.

The Membrane Filter (MF) method utilizes micropore filters through whichsamples are passed so that the bacteria are retained on the surface ofthe filter. This method is used most often when bacterial populationsare very small, and a large sample is needed to get adequate numbers.The filter is then placed on the surface of a chosen medium, incubated,and the bacterial colonies growing on the membrane filter surface arecounted and evaluated. This method is widely used and provides goodresults when combined with proper reagents and media A disadvantage ofthis method is that it is expensive and time-consuming. It also does notwork well with solid samples, or samples with high particulate counts.The MF method can be used in conjunction with the inventive methoddescribed in this application.

The m-Endo method is also used to determine the quantity of E. coli andgeneral coliforms and is an official USEPA approved method for testingwater quality. The medium is commonly used with a membrane filter and E.coli and general coliform colony forming units (CFU) grow as darkcolonies with a golden green metallic sheen. Due to a proven high rateof false positive error, typical colonies must be confirmed byadditional testing. Standard Methods for the Examination of water andWastewater, 20^(th) Edition, 9-10 &9-60 (1998).

Other tests, such as the Most Probable Number (MPN), utilize lactosecontaining broths (LST, BGLB, EC) to estimate numbers of generalcoliforms and E. coli, but have also been shown to have high rates orerror as well as being cumbersome and slow to produce results. Evans etal., Appl. Envir. Microbiol 41:130-138 (1981).

The reagent 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) isa known test compound for identifying coliforms. When acted on by theβ-galactosidase enzyme produced by coliforms, X-gal forms an insolubleindigo blue precipitate. X-gal can be incorporated into a nutrientmedium such as an agar plate, and if a sample containing coliforms ispresent, the coliforms will grow as indigo blue colonies. X-gal has theadvantage over the compound ONPG, described above, in that it forms awater insoluble precipitate rather than a diffusible compound, therebyenabling a quantitative determination of coliforms to be made when thetest sample is incorporated into or onto a solidified medium, or whencoliform colonies grow on the surface of a membrane filter resting on apad saturated with a liquid medium or on a membrane filter resting on asolid medium. Further, it does not require the use of ultraviolet light.

A similar compound, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc)is a known test compound for identifying E. coli. When acted on by theβ-glucuronidase enzyme produced by most E. coli, X-gluc forms aninsoluble indigo blue precipitate. X-gluc has the advantage over thecompound MUG, described above, in that it forms a water insolubleprecipitate, rather than a diffusible compound, thereby enabling aquantitative determination of E. coli to be made when the test sample isincorporated into or onto a solidified medium. X-gluc and its ability toidentify E. coli are described in Watkins, et al., Appl. Envir.Microbiol. 54:1874-1875 (1988). A similar compound,indoxyl-β-D-glucuronide, which also produces sharp blue colonies of E.coli, was described in Ley, et al., Can. J. Microbiol. 34:690-693(1987).

Although X-gal and X-gluc are each separately useful in the quantitativedetermination of either coliforms (X-gal) or E. coli (X-gluc), theseindicator compounds have the disadvantage that they each contain thesame chromogenic component. Therefore, they cannot be used together toidentify and distinguish both E. coli and general coliforms in a singletest with a single sample, since they both generate identically huedindigo blue colonies. A person using both reagents together would beable to quantitatively identify the total number of coliforms, the sameas if X-gal were used alone, but would not be able to indicate which ofthe colonies were E. coli and which were other coliforms besides E.coli.

A recently developed and highly commercially successful test method andtest medium for quantitatively identifying and differentiating generalcoliforms and E. coli in a test sample is described in U.S. Pat. Nos.5,210,022, and 5,393,662, both of which share common inventorship withthe present application and which are hereby incorporated by reference.This method and test medium improves upon prior art methods by allowingthe quantitative identification of general coliforms and E. coli in asingle sample. Additional confirmatory tests are not necessary. The testsample is added to a medium containing a galactosidase substrate, suchas 6-chloroindolyl-β-D-galactoside, and a β-glucuronidase substrate,such as 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc). Theβ-galactosidase substrate is capable of forming a water insolubleprecipitate of a first color upon reacting with β-galactosidase, and theβ-glucuronidase substrate is capable of forming a water insolubleprecipitate of a second color, contrasting with the first color, uponreacting with β-glucuronidase. As a result, general coliforms may bequantified by enumerating the colonies of the first color (havingβ-galactosidase activity), and E. coli may be quantified by enumeratingthe colonies of the second color (having both β-galactosidase andβ-glucuronidase activity). This technology has been widely copied.

Another recently developed test method and apparatus provides excellentresults for the differentiation and identification of general coliforms,E. coli and E. coli 0157 strains and non-coliform Enterobacteriaceae.The method and test medium are described in U.S. Pat. No. 5,726,031,which shares common inventorship with the present application, and whichis hereby incorporated by reference.

A certain class of substrates, referred to herein as “nonchromogenic,”have been used to detect various microorganisms. A dipslide fordetecting E. coli using hydroxy-quinoline-β-D-glucuronide is disclosedby Dalet et al., J. Clin. Microbiol, 33(5):1395-8 (1995). Similarly, atechnique for detection of E. coli in an agar-based medium using8-hydroxyquinoline-β-D-glucuronide is disclosed by James et al.,Zentralbl Bakteriol Mikrobiol Hyg [A], 267(3):316-21 (1988).

It is desirable to further improve the distinguishing colors generatedin the test media. That is to say, in prior art test media which detectand distinguishing two biological entities, confusion may arise betweenthe two colors which show in the media.

Further, it is desirable to be able to identify and differentiate otherclosely related organisms, such as members of the families Aeromonaceae,Vibrionaceae, and Salmonella and Shigella spp. For example, the genusAeromonas is closely related to coliforms and gives an almost identicalbiochemical test pattern. Further, the genus Vibrio is also an importanttype of bacteria that grows under the same general conditions ascoliforms. It is known to distinguish Aeromonas colonies from generalcoliforms by testing all colonies in a given sample for the presence ofcytochrome oxidase. Undesirably, however, this requires another set oftests. Further, Aeromonas is common in water and food, and it iscommonly indicated in test samples as general coliforms, which resultsin high a false positive error for general coliforms by most currenttest methods. The Aeromonas can be prevented from interfering with thecoliform results by adding certain antibiotics to the medium. However,the antibiotic amounts added must be carefully controlled. Further, theantibiotics which have been conventionally used have short life spans inthe media so that they lose their potency quickly in other than a frozencondition. It may often be desirable to be able to culture, identify andenumerate Aeromonas spp. which cannot be done if they are inhibited.

Further, in those cases where it is desirable to inhibit Aeromonas, itis desirable for a better method of so doing, one in which the shelflife of the medium is not appreciably reduced by the inclusion of aninhibitor.

Additionally, it is also desirable to distinguish strains of Salmonellaand Shigella from E. coli, general coliforms and Aeromonas.

SUMMARY OF THE INVENTION

The present invention overcomes the disadvantages of prior art methodsby providing a test method and medium for quantitatively orqualitatively identifying and differentiating biological entities in atest sample that may include a plurality of different biologicalentities.

The present invention introduces the use of “nonchromogenic” substratesto enhance the distinction among multiple colors produced by distinctbiological entities present in the inventive test medium. Unexpectedly,it has been discovered that other “chromogenic” substrates present inthe inventive test medium do not interfere with the substantially blackcolor achieved with the nonchromogenic substrate. That is to say, solong as a given biological entity is responsive to the nonchromogenicsubstrate, aggregations thereof present in the test medium will show asa substantially black color-independent of whether such biologicalentity is responsive to one, two or more chromogenic substrates whichare also present in the medium. The present invention exploits thishitherto unexplored property of nonchromogenic substrates.

In one form thereof, the present invention provides a test medium fordetecting, identifying and qualifying or quantifying first and secondbiological entities. The test medium includes a nutrient base mediumincluding ions of a salt, a chromogenic substrate and a nonchromogenicsubstrate. The first biological entity is responsive to thenonchromogenic substrate whereas the second biological entity isresponsive to the chromogenic substrate. In this test medium,aggregations of the first biological entity present in the test mediumare substantially black and aggregations of the second biological entitypresent in the test medium are a second color, the second color beingdistinguishable from the substantially black.

In a preferred form, the inventive test medium accounts for the firstbiological entity being responsive to the chromogenic substrate inaddition to the nonchromogenic substrate. In such event, aggregations ofthe first biological entity present in the test medium will nonethelessshow as substantially black.

Significantly, even though the aggregations of the first biologicalentity are responsive to both the first and second substrates in thepreferred form, these aggregations still show as substantially black inthe test medium. That is, the chromogenic substrate does not interferewith the substantially black color. Advantageously, this property ofnonchromogenic substrates allows several different biological entitiesto be identified and differentiated in a single medium, aggregations ofeach biological entity having a visually distinguishable color.

In another preferred form of the above-described inventive medium, themedium further includes the antibiotic nalidixic acid to inhibit thegrowth of Aeromonas, spp. Advantageously, it has been found thatnalidixic acid, as compared with cefsulodin, does not significantlyreduce the shelf life of the test medium incorporating it.

In this connection, another form of the present invention provides amethod of making a test medium for detecting at least one first type ofbiological entity and inhibiting a second type of biological entity fromgrowing in the medium. The method includes the steps of combiningdesired substrates with a nutrient base medium; adding an inhibitor tothe medium; and then sterilizing the medium by subjecting the medium toat least 100° C. Because the inhibitor is added as an initial step,subsequent sterile addition of inhibitor is unnecessary.

In another form thereof, the present invention provides a test mediumfor detecting, identifying and qualifying or quantifying first, secondand third biological entities. The test medium includes a nutrient basemedium including ions of a salt. First and second chromogenic substratesand a nonchromogenic substrate are provided in the test medium. Thefirst and second biological entities are responsive to the first and thesecond chromogenic substrates, respectively, and the third biologicalentity is responsive to the nonchromogenic substrate. Aggregations ofthe first biological entity present in the test medium are a firstcolor, aggregations of the second biological entity present in the testmedium are a second color, and aggregations of the third biologicalentity present in the test medium are substantially black.

In a preferred form, the inventive test medium accounts for the thirdbiological entity being responsive to the first and/or the secondchromogenic substrates in addition to the nonchromogenic substrate. Insuch event, aggregations of the third biological entity will nonethelessshow as substantially black.

It should be appreciated that the use of a nonchromogenic substratealong with one or more chromogenic substrates synergistically increasesthe number of biological entities that can be detected and distinguishedin a single medium and synergistically increases the possible colorcombinations for a given set of biological entities to be detected.Stated another way, including a nonchromogenic component as one of thesubstrates synergistically increases the degrees of freedom in selectingother substrates and corresponding colors for a test medium. This is sobecause an aggregation of the biological entity which is responsive tothe nonchromogenic substrate will dependably show as substantiallyblack. No combined color effects need be accounted for with thenonchromogenic substrates. For example, in a test medium including threechromogenic substrates and a nonchromogenic substrate, at least threecombined color combination effects are avoided by using the onenonchromogenic component, as compared with using four chromogeniccomponents.

The present invention, in another form thereof, provides a test mediumcapable of detecting, quantifying, and differentiating general coliformsand/or E. coli spp. under ambient light. The test medium comprises anutrient based medium including a salt. A first substrate capable offorming a first water insoluble component of a first color in thepresence of E. coli and the ions of the salt is provided in the medium.The first color is substantially black. A second substrate capable offorming a second water insoluble component of a second color in thepresence of general coliforms is provided. The second color is visuallydistinguishable from the first color. Thus, colonies of E. coli presentin the test medium are indicated by the first substantially black colorand colonies of general coliforms are indicated by the second color.

In a preferred form of the above invention, the test medium furtherincludes a third substrate capable of forming a third water insolublecomponent of a third color in the presence of Salmonella or Shigellaspp. The third color is distinguishable from the first and secondcolors, whereby the test medium is capable of quantifying and/ordifferentiating E. coli, general coliforms and Salmonella or Shigellaspp. Further, the substrates are selected such that general coliformspresent in the test medium will also react with the third substrate toform a water insoluble component which includes the third color.Consequently, general coliform colonies are indicated in the test mediumas a fourth color, the fourth color being a combination of the secondcolor and the third color. The fourth color is visually distinguishablefrom the first and third colors. Still further, the substrates can beselected such that Aeromonas spp. form an insoluble component of thesecond color by reacting with the second substrate, but not the firstand third substrates. Thus, in the inventive test medium, E. colicolonies will be generally black, general coliform colonies will be thefourth color, Aeromonas colonies will be the second color and Shigellaor Salmonella colonies will be the third color.

In another form thereof, the present invention provides a method fordetecting, quantifying and differentiating under ambient light generalcoliforms, E. coli, and at least one of the genera Aeromonas, Salmonellaor Shigella in a test sample. The method comprises the steps ofproviding a nutrient base medium including first, second and thirdsubstrates. Each of the substrates is capable of forming a waterinsoluble component in the presence of at least one of generalcoliforms, E. coli Aeromonas, Salmonella or Shigella. The substrates areselected such that colonies of E. coli produced in the test medium are afirst color, colonies of general coliforms produced in the test mediumare a second color, and colonies of one of Aeromonas and Salmonella orShigella produced in the test medium are a third color. Each of thecolors are visually distinguishable. The test medium is inoculated withthe test sample and then incubated. The test medium is then examined forthe presence of first colonies having the first color, second colonieshaving the second color, and third colonies having the third color. Thefirst colonies are E. coli, the second colonies are general coliforms,and the third colonies are one of Aeromonas, Salmonella or Shigella.

In a preferred form thereof, the inventive method further includesadding ions from a salt to the test medium to react with one or more ofthe substrates. In so doing, a precipitate is produced which shows as asubstantially black color in the presence of the specific enzyme forthat substrate. A preferred compound for forming the substantially blackcolor in the presence of the ions of the salt consists of aβ-D-glucuronide. These compounds release an aglycone when hydrolizedwhich forms a substantially black insoluble complex in the presence ofions.

In another preferred form of the inventive method, the method furthercomprises examining the test medium for the presence of fourth colonieshaving a fourth color, wherein the substrates are selected such thatcolonies of Aeromonas are the third color and colonies of Salmonella orShigella are the fourth color, the fourth color being visuallydistinguishable from the first, the second and the third colors. Morepreferably, the substrates are selected such that the first color issubstantially black, the second color is substantially blue-violet, thethird color is substantially red-pink and the fourth color issubstantially teal-green.

In another preferred form of the inventive method, the substrates areselected such that colonies of Aeromonas as well as colonies ofPlesiomonas and Vibrios are indicated as the third color.

One advantage of the present invention is that it uses a nonchromogenicsubstrate along with one or more chromogenic substrates and therebysynergistically increases the degrees of design freedom in selectingcolors for the inventive test medium. This is so because the chromogenicsubstrates do not interfere with the substantially black precipitateformed by the nonchromogenic substrate.

Another advantage of the present invention is that enables thequantification, identification and differentiation of four (4) differentbacterial strains simultaneously in a single test medium using a singletest sample, under ambient lighting. Subsequent tests with theirconcomitant extra time spent and extra costs are avoided. Of course, theinventive test medium of the present invention could also be used purelyfor qualitative purposes, as a mere presence/absence (P/A) test.

Yet another advantage of the present invention is that the substratesare selected such that the colors are easy to visually distinguish fromone another without the need for UV light or other visual aids, otherthan, perhaps, magnification means. For example, in a preferredembodiment, E. coli colonies are clearly indicated by a precipitatehaving a substantially black color, general coliform colonies areindicated by a blue-violet color, Aeromonas colonies are indicated by ared-pink color, and Salmonella or Shigella colonies are indicated by ateal-green color. Because these colors are visually so distinct,confusion among the colors is greatly reduced as compared to prior artmedia.

Another advantage of the test medium of the present invention is itsflexibility and ease of use. The incubation temperature is not criticalas growth and differentiation of the organisms mentioned may occurwithin an optimum range. Therefore, resuscitation steps are avoided andinhibition of temperature sensitive strains does not occur. Also,inexpensive equipment may be used.

Yet another advantage of the present invention is that it intensifiesthe color distinction obtained in a test medium for identifying anddifferentiating E. coli from general coliforms. In a preferred testmedium, E. coli colonies present a substantially black color, whereasgeneral coliforms present a red-pink color, the distinction therebetweenbeing much more apparent than in prior art test media. Confusion betweenthe two colors is therefore greatly reduced.

Still another advantage of the present invention is that it enables theidentification and differentiation of Aeromonas spp. from generalcoliforms. Prior art test media undesirably require using a cefsulodininhibitor for preventing Aeromonas spp. from growing therein. However,the use of cefsulodin as an inhibitor requires an extra step in theprocess, viz., sterile addition of filter sterilized antibiotic, and isdifficult to control. Further, the presence of cefsulodin significantlyreduces the effective shelf life of the medium. Further, the use of aninhibitor, obviously, prevents the detection and quantitification ofAeromonas spp. Advantageously, with the present invention, Aeromonasspp. can be detected, quantified and differentiated from generalcoliforms in a single medium.

As a related advantage, if it is nonetheless desired to inhibit coloniesof Aeromonas spp. from growing in the test medium, the present inventionprovides a superior means for doing so. Specifically, preferred forms ofthe present invention employ nalidixic acid as an inhibitor, which hasbeen shown to have a far less deleterious effect to the shelf-life ofthe medium incorporating it. Further, nalidixic acid can be added aspart of the initial medium formulation prior to sterilization, therebyavoiding a costly and difficult process step which is required withcefsulodin. Finally, nalidixic acid is much less expensive thancefsulodin.

Another advantage of the present invention is that it can provide a testmedium for qualitative or quantitative testing. That is, the test mediain accordance with the present invention can be used as merepresence/absence test devices, or can be used to quantify variousbiological entities showing as different colored colonies on theinventive test media.

DETAILED DESCRIPTION OF THE INVENTION

The method and medium of the present invention allow the simultaneousdetection, quantification, identification and differentiation of avariety of selected biological entities in a sample of mixed populationsof biological entities. The inventive method and medium are particularlyuseful for the detection, quantification, identification anddifferentiation of E. coli and general coliforms, and furtherquantitative identification and differentiation of other selectedbiological entities, including Aeromonas, Salmonella, Shigella,Pseudomonas, and Vibrio bacterial species.

The method and test media incorporating the present invention utilizethe fact that the enzymatic activity of biological entities andspecifically of bacteria varies with the genus, and/or family ofbacteria of interest. The method and test media incorporating thepresent invention further utilize the fact that various enzymeidentifying substrate complexes can be used to identify specific enzymeswith the production of distinctive colors. Significantly, the method andtest media incorporating the present invention exploit the fact thatchromogenic substrates present in a test medium do not interfere withthe substantially black color produced by nonchromogenic substrates.

While nonchromogenic substrates are known in the art, per se, theirdistinct properties vis-á-vis chromogenic substrates have beenunrecognized. However, the behavior of a nonchromogenic substrate in amedium including a combination of chromogenic substrates is unique. Toillustrate, aggregations of a biological entity which is responsive totwo chromogenic substrates will typically show in a test medium as acombination of the the two colors produced upon cleavage of the tworespective substrates. If three chromogenic substrates are involved, thecombined color effect could be prohibitively difficult to predict andaccount for. Further, inherent variations in the amount of enzymesproduced by particular strains of biological entities can result indifferent shades or hues of colors upon cleavage of the chromogenicsubstrates. Consequently, the colors can be difficult to distinguish forthe lay person examining the test medium. Chromogenic substrates musttherefore be chosen in view of the other chromogenic substrates plannedfor inclusion in a given test medium.

Such is not the case with the nonchromogenic components. Whileaggregations of biological entities which are responsive to chromogenicsubstrates in addition to nonchromogenic substrates may show in the testmedium as having a colored or fluoroescent “halo,” such aggregationsnonetheless appear substantially black and are therefore easy toidentify. Unlike chromogenic substrates, multiple “degrees of freedom”are achieved with the nonchromogenic components by not having to takeinto account combined color effects.

Using a nonchromogenic substrate enables a single test medium todifferentiate four (4) different bacterial strains with four (4)visually distinguishable colors. The black color is superior in that itis difficult to mistake. Further, the substantially black pigmentationdoes not diffuse so that the location of the colonies is precisely knownand the colonies can be accurately counted. The nonchromogenicsubstrates produce an insoluble chelated compound which is differentthan the dimer which is produced by the chromogenic substrates.

The inventive test medium and method allows not only a detection,quantification or qualitative identification and differentiation ofgeneral coliforms and E. coli, but also of Salmonella, Shigella andAeromonas, as well as Plesiomonas and Vibrio. Plesiomonas and Vibriosspecies are determined but not differentiated from Aeromonas species asthey are very closely related.

Definitions

Biological entities, such as general coliforms, E. coli., etc., areherein referred to as being “responsive” to certain chromogenic andnonchromogenic substrates. More specifically, a biological entity willpredictably produce specific enzymes when the entity is present in atest medium such as the one described hereinbelow. These enzymes willselectively cleave chromogenic and nonchromogenic substrates. Uponcleavage, these substrates produce a color in the test medium. Themechanism for producing the color is different for chromogenic andnonchromogenic substrates, as described hereinbelow.

Microorganisms having β-galactosidase activity include those commonlyknown by the designation “coliform.” There are various definitions of“coliform,” but the generally accepted ones include bacteria which aremembers of the Enterobacteriaceae family, and have the ability toferment the sugar lactose with the evolution of gas and acid. Mostcoliforms are positive for both α- and β-galactosidase. That is, theyproduce both α- and β-galactosidases.

Microorganisms having β-glucuronidase activity in addition togalactosidase activity primarily include most strains of Escherichiacoli. That is, E. coli is positive for both α- and β-galactosidase aswell as β-glucuronidase.

The term “general coliforms” as used in this application refers tocoliforms other than the various strains of E. coli. These “generalcoliforms” are gram-negative, non-spore forming microorganisms generallyhaving α- and β-galactosidase activity (i.e., lactose fermenters), butnot having β-glucuronidase activity, and having the ability to fermentthe sugar sorbitol.

For purposes of this specification, a “chromogenic substrate” is asubstrate which needs no additional chemicals present in the test mediumupon hydrolysis for color production. That is, a chromogenic substrateis cleaved by the specific enzyme corresponding to that substrate toform a dimer with the color being concentrated in the area of cleavageof the substrate. Many chromogenic substrates are known in the art. Forpurposes of this specification “chromogenic” includes fluorogenicsubstrates. The products of fluorogenic substrates require ultraviolet(UV) light to be detected and are more soluble than preferredchromogenic substrates, so are therefore generally not preferred for usewith the test media disclosed hereinbelow.

Certain substrates, referred to herein as “nonchromogenic,” produce adark, substantially black precipitate in the presence of ions of a saltand enzyme activity. For example, 8-hydroxyquinoline-β-D-glucuronide,when included in a medium along with a salt that produces ions, such asferric ammonium citrate, will produce a substantially black precipitatein the presence of β-glucuronidase produced by E. coli or otherbiological entities. More specifically, upon cleavage of thenonchromogenic substrate by the particular enzyme, a substantially blackwater-insoluble complex forms in the medium. The substantially blackprecipitate consists of the ferric ions and the aglycone released whenthe substrate is hydrolized by the glucuronidase from E. coli. Thisprecipitate is a chelated compound which does not diffuse. Nor is thesubstantially black color susceptible to interference from chromogeniccompounds present in the test medium.

For purposes of this specification a “nonchromogenic substrate” meansthat a chemical in addition to those used with chromogenic componentsmust be present in the test medium when the substrate is cleaved by itscorresponding enzyme. The substantially black precipitate formed therebyis a combination of the substrate—salt complex and is not a dimer as isformed by the “chromogenic compounds.”

For purposes of this specification, the expression “under ambient light”refers to the visible spectrum, i.e., colors which can be seen anddistinguished with the naked eye. A colony present in a test mediumwhich requires ultraviolet light to be seen, for example, would not fallunder the definition “under ambient light”. However, it is to beunderstood that the term “under ambient light” includes using amagnification device, if necessary. Magnification can be especiallyhelpful when counting numerous colonies. The term “visuallydistinguishable” refers to two or more colors which can bedifferentiated under ambient light.

For purposes of this specification, the term “substantially black”includes dark brown to black, and also includes black with variouscolored halos, such as red-violet, green, fluorescent, etc.

For further purposes of this specification, color names recited hereinare given as guidance, but it is to be understood that the color namesare to be read broadly. That is, there can be overlap among the recitedcolors. This is because, as discussed, biological entities producevarying amounts of enzymes, which in turn affects the shade or hue ofthe resulting color.

The term “β-galactosidase substrate” as used herein refers to aβ-galactoside comprising galactose joined by β-linkage to a substituentthat forms a water insoluble colored compound when liberated by theaction of β-galactosidase on the substrate. Similarly, the term“α-galactosidase substrate” as used herein refers to α-galactosidecomprising galactose joined by α-linkage to a substituent that forms awater insoluble colored compound when liberated by the action ofα-galactosidase on the substrate. The term “β-glucuronidase substrate”as used herein refers to a β-glucuronide comprising glucuronic acidjoined by β-linkage to a substituent that forms a water insolublecolored precipitate when liberated by the action of β-glucuronidase onthe substrate.

The α- and β-galactosidase substrates and compounds and any othersubstrates described herein as well as the β-glucuronidase substratesand compounds and any other substrates described herein may comprisecarboxylate salts formed by reacting a suitable base with theappropriate galactoside or glucuronic carboxyl group. Suitable basesinclude alkali metal or alkaline earth metal hydroxides or carbonates,for example, sodium hydroxide, potassium hydroxide, calcium hydroxide,magnesium hydroxide, and corresponding carbonates; and nitrogen basessuch as ammonia, and alkylamines such as trimethylamine, triethylamineand cyclohexylamine.

Designing a Test Medium for Specific Biological Entities

Certain members of the family Enterobacteriaceae can be distinguished bythe presence of α-galactosidase activity in the absence ofβ-galactosidase activity, or vice-versa. For example, most Salmonellaand Shigella spp. are positive for α-galactosidase, but negative forβ-galactosidase. Similarly, Aeromonas spp. can be distinguished fromother members of the family Enterobacteriaceae by the presence ofβ-galactosidase activity in the absence of α-galactosidase activity. Themethod and medium incorporating the present invention are designed totake advantage of these distinguishing characteristics. For example, thespecificity of enzyme activity for Salmonella and Aeromonas spp., asopposed to general coliforms, can be exploited, as illustrated below.

The method described herein is particularly suitable for the detection,quantification or qualitative identification and differentiation of thedifferent classes of microorganisms described previously, viz., generalcoliforms, E. coli, Aeromonas and Salmonella and Shigella spp. Althoughthe inventive method is particularly suitable for these particularmicroorganisms, it is not limited thereto. Instead, the techniquesdescribed herein have application to the identification anddifferentiation of a wide variety of biological entities.

That is, specific biological entities are “responsive” to varioussubstrates. More particularly, these biological entities predictablyproduce or contain known enzymes. Substrates, either chromogenic ornonchromogenic, can be selected which, in the presence of a particularenzyme(s), will form an insoluble component of a predictable anddistinguishable color. Multiple substrates can be selected tosimultaneously identify a plurality of distinct biological entities in asingle test medium, aggregations of each distinct entity beingidentifiable by a separate, distinguishable color. Further, while thepreferred embodiments disclosed herein distinguish all of the variousaggregations present in a test medium under ambient light, as that termis defined herein, such is not necessary. For example, severalsubstrates disclosed herein require the use of ultraviolet light for theaggregations present in the medium to be seen.

Table I lists various enzymes whose presence may be detected usingcertain of the substrates listed in Table II.

TABLE I Enzymes and Abbreviations Aara = α-D-arabinopyranosidase Bglu =β-D-glucopyranosidase Agal = α-D-galactopyranosidase Bgluc =β-D-glucuronidase Aglu = α-D-glucopyranosidase Bman =β-D-mannopyranosidase Bcel = β-D-cellopyranosidase Bxyl =β-D-xylopyranosidase Bfuc = β-D-fucopyranosidase Nagal = N-acetyl-β-D-galactopyranosidase Bgal = β-D-galactopyranosidase Naglu = N-acetyl-β-D-glucopyranosidase Afuc = α-D-fucopyranosidase Bara =β-D-arabinopyranosidase Bxyl = β-D-xylopyranosidase Acel =α-D-cellopyranosidase Aman = α-D-mannopyranosidase Agluc =α-D-glucuronidase Axyl = α-D-xylopyranosidase Nagluc =N-acetyl-β-D-glucuronidase esterase

TABLE II Various Substrates and Color Upon Cleavage 6-chloro-3-indolylsubstrates Pink 5-bromo-4-chloro-3-indolyl substrates Teal 3-indolylsubstrates Teal N-methylindolyl substrates Green nitrophenyl substratesYellow nitroaniline substrates Yellow 8-hydroxyquinoline substrates (andion of salt) Substantially black cyclohexenoesculetin substrates (andion of salt) Substantially black esculetin substrates (and ion of salt)Substantially black quinoline substrates (and ion of salt) Substantiallyblack 5-Iodo-3-Indolyl substrates Purple 5-Bromo-6-Chloro-3-Indolylsubstrates Magenta 6-Fluoro-3-Indolyl substrates Pink coumarinsubstrates Fluorescent fluorescein substrates Fluorescent rhodaminesubstrates Fluorescent resorufun substrates Fluorescent

Specific substrate compounds applicable for use with the test medium ofthe present invention are available as follows:

5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) is acommercially available β-galactosidase substrate that produces aninsoluble precipitate having an approximately teal color when reactedupon by β-galactosidase and is available from Biosynth International,Naperville, Ill.

6-chloro-3-indolyl-β-D-glucuronide is a compound which produces aninsoluble precipitate having a magenta color, the preparation of whichis described in the aforementioned incorporated by reference U.S. Pat.No. 5,210,022 and is available from Research Organics, Cleveland, Ohio.

The compound 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) is acommercially available β-glucuronide that produces an insolubleprecipitate having an approximately teal color when reacted upon byβ-glucuronidase. Similarly, indoxyl-β-glucuronide is a similar compound,the preparation of which is described in the aforementioned article byLey et al., in Can J. Microbiol., the disclosure of which isincorporated by reference.

Another suitable β-galactoside is the compound6-chloro-3-indolyl-β-D-galactoside which produces an insolubleprecipitate having a pink/magenta color, the preparation of which isdescribed in the aforementioned U.S. Pat. No. 5,210,022.

Other suitable compounds applicable as substrates in the practice of thepresent invention are specified in U.S. Pat. No. 5,210,022, all of whichare incorporated herein by reference.

The substrate 8-hydroxyquinoline-β-D-glucuronide is a commerciallyavailable β-glucuronide that, in the presence of metallic ions such asiron, produces an insoluble precipitate having a substantially blackcolor when reacted upon by β-glucuronidase and in the presence of otherα- or β- galactoside substrates. 8-hydroxyquinoline-β-D-glucuronide isavailable from Biosynth International, Naperville, Ill.

Further, a salt providing ions suitable for use with the presentinvention is ferric ammonium citrate, available from Sigma Chemical, St.Louis, Mo. The cyclohexenoesculetin substrates are described in James etal., Appl. & Envir. Micro. 62:3868-3870 (1996) and in the presence offerric ions, produce an insoluble substantially black precipitate.

N-methyl-indolyl substrates such asN-methylhydroxy-β-D-galactopyranoside are commercially available fromBiosynth International, Naperville, Ill.

Nitrophenyl substrates, such as 2-nitrophenyl-β-D-galactopyranoside, arecommercially available from Biosynth International, Naperville, Ill.Similarly, nitroaniline compounds are available for synthesis throughSigma Chemical, St. Louis, Mo.

Other substrates producing a substantially black color include esculetinsubstrates such as cyclohexenoesculetin-β-D-galactoside, which isdescribed in James et al., Appl. & Envir. Microbiol. 62:3868-3870(1996). Quinoline substrates, such as8-hydroxyquinoline-β-D-galactopyranoside and8-hydroxyquinoline-β-D-glucuronide are available through BiosynthInternational, Naperville, Ill.

Iodo-indolyl substrates, such as 5-iodo-3-indolyl-β-D-galactopyranosideare available through Biosynth International, Naperville, Ill.

Several fluorescent substrates are suitable for use with the presentinvention. Coumarin substrates such as 4-methylumbelliferyl substratesand 5-trifluoromethylumbelliferyl substrates are commercially availablefrom Biosynth International, Naperville, Ill. Also suitable arefluorescein substrates, rhodamine substrates, and resorufin substrates.No commercial source is known for these three substrates but componentsare available from Sigma Chemical, St. Louis, Mo.

While specific examples of substrates suitable for use with the presentinvention have been enumerated hereinabove, such is not to be construedas limiting the invention in any manner. Instead, one of ordinary skillin the art can use Table IV and V hereinbelow to identify a virtuallylimitless number of substrates.

Preparation of Test Medium

The test medium is formed by combining the desired substrates with anutrient base medium. The nutrient base medium can be any culture mediumknown in the art for providing the maintenance and reproduction ofliving cells. Generally, such media include nutrients, buffers, water,and sometimes a gelling agent. Possible gelling agents include agars,pectins, carrageenans, alginates, locust bean, and xanthins, amongothers.

The following is an example of the preparation of a test medium suitablefor use in this invention. This example coincides with Example I, below.

The substrates 8-hydroxyquinoline-β-D-glucuronide,5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside, and6-Chloro-3-indolyl-β-D-galactopyranoside are added in quantities of 250mg/L medium; 70 mg/L medium; and 175 mg/L medium, respectively. Thesubstrates are added directly to the hot (75°-85° C.) medium (formulabelow) in a blender prior to sterilization.

Standard agar medium may be made by adding 15 gm of bacteriologicalquality agar gum to the following nutrient formula

Pancreatic Digest of Casein 5.0 gm Yeast Extract 3.0 gm DipotassiumPhosphate .3 gm Deionized Water 990 ml Ferric Ammonium Citrate 800 mg in10 ml deionized water (sterilized separately from the other components)

and then sterilizing at 121° C. for 15 minutes. The medium should beadjusted to result in a pH of 7.0. The sterilized agar medium is allowedto drop to a temperature of 45° C. in a water bath and then the sterilesolution containing the substrates prepared as described above is added.The medium is mixed thoroughly and poured into sterile petri plates at avolume of 20 ml/plate.

A pectin-based test medium may be prepared using the same stepsdescribed above except that 25 gm of low methoxyl pectin is used as thesolidifying agent and the medium is poured at room temperature intopetri plates containing a thin gel layer containing calcium ions whichcombine with the pectin to form a solid gel. A suitable pectin culturemedium is described in U.S. Pat. Nos. 4,241,186 and 4,282,317, thedisclosures of which are incorporated herein by reference. Apectin-based medium is preferred over a standard agar medium because ithas the advantages of convenience and temperature independence for theuser. The use of pectin media is well described and accepted as a resultof AOAC collaborative studies and other published and in-houseinvestigations.

A suitable pectin medium is commercially available from MicrologyLaboratories, LLC under the trademark Easygel®. Aqueous based mediumwithout gelling agent is available from Micrology Labs, Goshen Ind., foruse with membrane filters.

Inoculation of the Test Medium with the Sample

The test medium may be inoculated with a sample to be tested for thepresence of microorganisms by any method known in the art forinoculating a medium with a sample containing microorganisms. Forexample, the sample to be tested may be added to the petri plates priorto adding the nutrient base medium (pour plate technique) or spread onthe surface of the plates after they have cooled and solidified (swab orstreak plate technique). Liquid samples may also be filtered through amicropore (0.45 micrometer size) membrane filter which is then placed onthe surface of a solid medium or on a pad saturated with the medium.

Incubation of the Test Medium

The inoculated test medium is incubated for a sufficient time and atsuch a temperature for individual microorganisms present in the sampleto grow into detectable colonies. Suitable incubation conditions forgrowing microorganisms in a medium are known in the art. Commonly, thetest medium is incubated for about 24-48 hours at a temperature of about30°-40° C.

Unless inhibitors of the general microbial population are used, thegeneral microbial population as well as general coliforms, E. coli,Aeromonas spp., and Salmonella spp. and Shigella spp. will grow in theincubated test medium. Because the precipitates formed are insoluble inthe test medium, they remain in the immediate vicinity of microorganismsproducing the various enzymes. As the microorganisms reproduce to formcolonies, the colonies show as colony forming units having the colorproduced by the particular substrate.

For example, E. coli produces β-galactosidase and α-galactosidase, but,unlike general coliforms and Aeromonas spp., also producesβ-glucuronidase. Therefore, insoluble precipitates of each of theβ-galactosidase substrate, the α-galactosidase substrate and theβ-glucuronide substrate are formed by the action of the respectiveenzymes such that colonies of E. coli show as a substantially blackcolor, sometimes having a violet-blue halo therearound.

General coliforms produce β-galactosidase and α-galactosidase andconsequently cleave both the α-galactosidase and β-galactosidasesubstrates. In the present example, the5-Bromo-4-chloro-3-indolyl-α-D-galactoside substrate produces ablue-green or teal color, whereas the 6-Chloro-3-indolyl-β-D-galactosideproduces a pink, or red-pink color. Thus, general coliform colonies willshow as a blue-violet color, which is a combination of the colorsproduced by each of the α- and β-galactosides, respectively.

Significantly, however, it has been found that Aeromonas spp., which areclosely related to coliforms, and give an almost identical biochemicaltest pattern, are β-galactosidase positive and α-galactosidase negative.That is, Aeromonas spp. will not hydrolize the α-galactoside substrate.Therefore, Aeromonas colonies present in the test medium will show ascolonies having the pink-red color produced by the β-galactosidesubstrate.

Further, it has been found that most members of the genera Salmonellaand Shigella are positive for α-galactosidase, but negative forβ-galactosidase. That is, Salmonella and Shigella will not hydrolize theβ-galactosidase substrate. Therefore, colonies of Salmonella andShigella present in the test medium will appear as a teal, or blue-greencolor produced by the α-galactoside substrate. Occasionally, Shigellacolonies will appear black with a blue-green halo since some strains ofShigella are positive for β-glucuronidase, and some strains of Shigellawill appear blue/purple since some unusual strains are positive for bothα-galactosidase and β-galactosidase.

Examination of the Test Medium and Enumeration of Microorganisms

The substrates selected for the above example produce three distinctcolors, and general coliforms are indicated by a fourth color which is acombination of two of the three colors. That is, E. coli colonies showas substantially black, general coliform colonies show as blue-violet,Aeromonas colonies show as red-pink, and Salmonella and Shigellacolonies show as teal-green. While the individual shades of these colorsmay vary somewhat in the test medium due to factors such as varyingenzyme production of the biological entities, it has been found thatthese four colors are distinct enough so that confusion amongst them isunlikely.

The colonies of each type of microorganism may be enumerated by countingthe colonies or by other methods known in the art for enumeratingmicroorganisms on a test plate. The number of colonies of each typeindicates the number of microorganisms of each type originally presentin the sample before incubation.

Optional Ingredients Inhibitors

The method of the present invention does not require inhibitors.However, the medium may be made more selective for general coliforms andE. coli if desired by the addition of various compounds that areinhibitory to the general microbial population, but have little or noeffect on coliforms. Following are some compounds which may be used: a)bile salts, about 0.3 g/liter, b) sodium lauryl sulfate, about 0.2g/liter, c) sodium desoxycholate, about 0.2 g/liter, d) Tergitol 7,about 0.1 ml/liter. The use of one or more of these compounds reducesthe background (non-coliform) microorganism presence and makes a lesscluttered plate and eliminates the possibility of inhibition orinterference by the non-coliform organisms in the sample. The use ofcertain antibiotics may accomplish the same result.

Cefsulodin is commonly used in currently available test media to inhibitAeromonas spp. However, the use of cefsulodin as an inhibitor requiresan extra step in the process, viz., sterile addition of filtersterilized antibiotic. This step is difficult to control. Further, thepresence of cefsulodin significantly reduces the effective shelf life ofthe medium. It has been found that Nalidixic acid can be used instead ofCefsulodin to inhibit Aeromonas spp. with about the same efficacy.Nalidixic acid is preferable because it can survive the approximately120° C. temperature reached in autoclaving the test media. Therefore,unlike cefsulodin, nalidixic acid can be added to the test media as partof the initial media formulation prior to sterilization (see,preparation of test medium, above). It also follows that the resistanceof the nalidixic acid to unfavorable environmental conditions willresult in a longer shelf life for a medium containing it as compared tocefsulodin.

Inducers

It is possible that the enzyme production of the general coliforms maybe enhanced by the addition to the medium formulations of very smallamounts of substances known as enzyme inducers. One specific inducer forβ-galactosidase is available and is known chemically asisopropyl-β-thiogalactopyranoside. Adding approximately 100 mg/liter ofmedium has a positive and noticeable effect on the speed of enzymeproduction for some species of coliforms. Other enzyme inducers areavailable and may be added to media formulations if enhanced enzymeproduction is deemed helpful.

EXAMPLES

Listed below are broad examples of test media enzyme substratecombinations to be used in combination with the nutrient formuladiscussed above or other suitable nutrient formulas which may beprepared in practicing the present invention.

Table III illustrates the flexibility of the preferred embodimentsincorporating the present invention. Table III is a matrix of some ofthe possible four-color combinations available for the preferredbiological entities E. coli, general coliforms, and at least one of thegenera Aeromonas, Salmonella or Shigella to be detected by using theteachings of this disclosure. Other color combinations are possible. Inmany cases, a plurality of different substrates will achieve a desiredresult, the only difference being the colors detected for a specificenzyme. The preferred color choice for the detection of E. coli isdenoted with an asterisk in Table III, depending on the colors chosen todetect other microorganisms. As discussed above, the substantially blackcolor is preferred because other chromogenic substrates do not interferewith it and the substantially black color is easy to distinguish fromthe other colors.

As discussed above, the use of Table III requires taking into accountthe combined color effect discussed above which is produced by theinclusion of multiple chromogenic substrates in a single medium. Forexample, with reference to the first entry in Table III, it can beunderstood that general coliforms will appear as a combination of (1)red-pink (magenta) and (2) teal, the resulting color being blue-violet.This is the case because general coliforms are responsive to twochromogenic substrates. Similarly, general coliforms will show in a testmedium in accordance with the second entry of Table III as a combinationof (1) red-pink (magenta) and (2) yellow.

TABLE III Color possibilities for detection of preferred microorganismsdesired red-pink or color mageneta teal green yellow black fluorescentfluorescent fluorescent 1 general general E. coli E. coli E. coli* E.coli coliforms coliforms Aeromonas Salmonella/shi- gella 2 general E.coli E. coli general coliforms E. coli* E. coli coliforms Salmonella/Aeromonas shigella 3 E. coli general E. coli general coliforms E. coli*E. coli coliforms Salmonella/ Aeromonas shigella 4 general E. coligeneral E. coli E. coli* E. coli coliforms coliforms Salmonella/shi-Aeromonas gella 5 E. coli general general E. coli E. coli* E. colicoliforms coliforms Salmonella/shi- Aeromonas gella 6 E. coli E. coligeneral general coliforms E. coli* E. coli coliforms Salmonella/Aeromonas shigella 7 E. coli E. coli E. coli E. coli general generalcoliforms E. coli coliforms Salmonella/ Aeromonas shigella 8 general E.coli E. coli E. coli E. coli* general coliforms E. coli coliformsSalmonella/ Aeromonas shigella 9 E. coli general E. coli E. coli E.coli* general coliforms E. coli coliforms Salmonella/ Aeromonas shigella10 E. coli E. coli general E. coli E. coli* general coliforms E. colicoliforms Salmonella/ Aeromonas shigella 11 E. coli E. coli E. coligeneral coliforms E. coli* general coliforms E. coli AeromonasSalmonella/ shigella 12 general E. coli E. coli E. coli general E. colicoliforms coliforms Aeromonas Salmonella/shi- gella 13 E. coli generalE. coli E. coli general E. coli coliforms coliforms AeromonasSalmonella/shi- gella 14 E. coli E. coli general E. coli general E. colicoliforms coliforms Aeromonas Salmonella/shi- gella 15 E. coli E. coliE. coli general coliforms general E. coli Aeromonas coliformsSalmonella/shi- gella 16 E. coli E. coli E. coli E. coli E. coli* E.coli general general coliforms coliforms Aeromonaceae Salmonella/ monasshigella *= preferred color for E. coli

Table IV is a partial list of enzyme patterns for biological entitiespreferred to be to be detected in accordance with the teachings of thisdisclosure. It is to be understood that one of ordinary skill in the artwould readily recognized that other enzymes which are known and havebeen produced, and enzymes which are known only on a theoreticallylevel, would also perform satisfactorily.

TABLE IV GENERAL Salmonella/ ENZYME NAME E. coli COLIFORM AeromonasShigella Plesiomonas Vibrio Aara = + + − + α-D-arabinopyranosidase Agal= + + − + α-D-galactopyranosidase Aglu = − + + − + α-D-glucopyranosidaseBcel = − + − − − − β-D-cellopyranosidase Bfuc = + + + − − −β-D-fucopyranosidase Bgal = + + + − + + β-D-galactopyranosidase Bgal= + + + − − − β-D-glucopyranosidase Bgluc = + − − − − −β-D-glucuronidase Bman = + + − + + + β-D-mannopyranosidase Bxyl = − + −− βD-xylopyranosidase Nagal = − + + − + +N-acetyl-β-D-galactopyranosidase Naglu = − + + − + +N-acetyl-β-D-glucopyranosidase Aman = − − − − − − α-D-mannopyranosidaseesterase = − − − + − − esterase

Table V is a matrix which teaches a wide variety of substrates and theirassociated colors for use in test media in accordance with the teachingsof this disclosure. The left hand side of Table V indicates the colorthat will result when the listed chromogenic component is cleaved fromits corresponding substrate by the specific enzyme for that substrate.In the case of the nonchromogenic components, the color is substantiallyblack and the reaction mechanism requires the presence of ions of a saltupon cleavage of the substrate, as explained above.

Test enzymes which are produced by certain biological entities (seeTable IV) are found at the right hand side of table V. “Substratecomponents” are shown to the left of the specific test enzymes. Each ofthe substrate components listed on the right hand side of table V can becombined with any of the chromogenic or nonchromogenic components listedon the left hand side of table V to identify a specific substrate foruse in a test medium. It can therefore be understood that Table Vteaches a large quantity of substrates possible for use in accordancewith the present invention. Many of the substrates identified by theabove-described use of table V are commercially available, whereas themethod for producing other identified substrates is described in theliterature. Still other substrates identified by using table V are onlytheoretically possible.

Nonchromogenic components are included at the bottom left hand side ofTable V, and are different from the chromogenic components because theydo not form specific colors upon cleavage. Instead, the quinoline oresculetin components combine with ions of a salt (e.g., ferric salt)which must be present in the medium when the substrate is cleaved by thespecific enzyme. The substantially black precipitate formed by thenonchromogenic components is a combination of the quinoline oresculetin—iron complex rather than a dimer which is formed by thechromogenic components.

Unlike nonchromogenic components, the chromogenic components should beselected in view of all other chromogenic components selected for themedium and in view of the enzyme patterns of the entities to bedetected. The selection of chromogenic components should maximize thedistinction among the respective colors produced.

While many various chromogenic component and substrate component/enzymepossibilities are taught by Table V, other possibilities within thescope of the appended claims would be possible by one of ordinary skillin the art. For example, as shown in Table V, one of ordinary skill inthe art could combine an N-acetyl group with many of the sugars of thesubstrate components listed in Table V. For example, an N-acetyl groupcould be combined with β-D-mannopyranoside to formN-acetyl-β-D-mannosaminide, the corresponding enzyme beingN-acetyl-β-D-mannosaminidase. Any of the chromogenic components ornonchromogenic components listed on the left hand side of Table V couldthen be combined with the substrate component to identify a substrate.If the substrate is commercially available or the method of making it isknown, the substrate could be used in a test medium. Upon cleavage ofthe substrate by the corresponding enzyme in the test medium, the colorlisted will appear.

Generally, the teachings of this disclosure can be used as follows tomake a test medium for detecting various microorganisms or cell types.First, the microorganisms desired to be detected and differentiated areselected. The preferred organisms to be detected are E. coli, generalcoliforms, and at least one of the genera Aeromonas, Salmonella orShigella. Enzymes produced by the selected organisms can be identifiedwith reference to Table IV. Equipped with knowledge of specific enzymesproduced by each microorganism, one can then identify correspondingsubstrates components from the right hand side of Table V. Dependingupon the color desired, one can select a chromogenic or nonchromogeniccomponent from Table V to be combined with the substrate component toidentify a substrate for inclusion in the test medium. If the substratethereby identified is commercially available or the method of itssynthesis is known, the substrate can be used in the test medium.

TABLE V COLOR COMPONENT AND SUBSTRATE COMPONENT MATRIX CHROMOGENICCOMPONENT & (COLOR) 6-fluoro-3-indolyl- (pink) 6-chloro-3-indolyl-(pink/red) 5-bromo-6-chloro-3-indolyl- (magenta) 3-indolyl- (teal)5-bromo-4-chloro-3-indolyl- (teal) 5-iodo-3-iondolyl- (purple)N-methylindolyl- (green) 4-methylumbelliferyl- (fluorescent) rhodamine-(fluorescent) fluorescein- (fluorescent) resorufin- (fluorescent)coumarin (fluorescent) nitrophenyl- (yellow) nitroaniline (yellow)NONCHROMOGENIC COMPONENT (COLOR) 8-hydroxyquinoline plus ions-(substantially black) 3,4-cyclohexenoesculetin plus ions (substantiallyblack) esculetin plus ions- (substantially black) SUBSTRATECOMPONENT - - TEST ENZYME α-D-arabinopyranoside -- Aara.α-D-cellopyranoside -- Acel. α-D-fucopyranoside -- Afuc.α-D-galactopyranoside -- Agal. α-D-glucuronide -- Agluc.α-D-mannopyranoside -- Aman. α-D-xylopyranoside -- Axyl.β-D-arabinopyranoside -- Bara. β-D-cellopyranoside -- Bcel.β-D-fucopyranoside -- Bfuc. β-D-galactopyranoside -- Bgal.β-D-glucopyranoside -- Bglu. β-D-glucuronide -- Bgluc.β-D-mannopyranoside -- Bman. β-D-xylopranoside -- Bxyl.N-acetyl-β-D-galactosaminide -- Nagal N-acetyl-β-D-glucosaminide --Naglu N-acetyl-β-D-glucuronaminide -- Nagluc N-acetyl + other sugarcomponents butyrate -- esterase caprylate -- esterase palmitate --esterase

Tavle VI is a concise summary of the specific examples.

TABLE VI EXAMPLE SUMMARIES General Salmonella/ Example # Substrate E.coli Coliforms Aeromonas Shigella I 8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color BlackPurple-Blue Pink Teal II 8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-mannoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color BlackPurple-blue Red-pink Purple-blue IIIA 8-hydroxyquinoline-β-D-glucuronideX 6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color BlackPurple-blue Purple-blue Pink IIIB 8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color BlackPurple-blue Purple-blue Teal IIIC May eliminate Aeromonas withinhibitors which allows removal of 6-chloro-3-indolyl-β-D-galactopyranoside from Examples IIIA and IIIB IV6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-mannoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color Purple-Purple-blue Pink Purple-blue Blue V-A6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color Purple-Purple-blue Purple-blue Pink blue V-B6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color Purple-Purple-blue Purple-blue Teal blue V-C May eliminate Aeromonas withinhibitors which allows removal of substrate No. 1 from example V-A andallows removal of substrate No. 3 from example V-B VI5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-galactopyranoside X X X color Purple- Purple-bluePink Teal blue VII 8-hydroxyquinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-N-acetyl-β-D-galactosaminide X X color BlackPurple-blue Pink Teal Note: In example 7, Vibrio and (see note)Plesiomonas also show as pink along with Aeromonas VIII8-hydroxyquinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-mannoside X X X6-chloro-3-indolyl-N-acetyl-β-D-galactosaminide X X color BlackPurple-blue Pink Purple-blue Note: In example 8, Vibrio (see note) andPlesiomonas also show as pink along with Aeromonas IX5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-N-acetyl-β-D-galactopyranoside X X color Purple-Purple-blue Pink Teal blue X6-chloro-3-indolyl-N-acetyl-β-D-galactopyranoside X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-galactopyranoside X X X color Purple- Purple-bluePink Teal Note: For example 10, Vibrio blue (see note) and Plesiomonasalso show as pink along with Aeromonas XI8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X (or)5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside X X X color Black PinkPink Not detected Note: In example 11, Aeromonas or or may be eliminatedby adding Teal Teal inhibitors XII8-hydroxyquinoline-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X (or)6-chloro-3-indolyl-α-D-galactopyranoside X X X color Black Black BlackTeal Note: In example 13, Aeromonas or may be eliminated by adding anPink inhibitor XIII Use same substrates as in example No. 1, and add:Enterobacter and Klebsiella showing4-methyl-umbelliferyl-β-D-xylopyranoside as black colonies willfluoresce, thereby allowing reduction in false positive count of E.coli. XIV 8-hydroxy-quinoline-β-D-glucuronide X6-chloro-3-indolyl-caprylate X color Black Red-pink XV8-hydroxy-quinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-caprylate X6-chloro-3-indolyl-α-D-galactopyranoside X X X color Black Red-pinkBlue-violet XVI 8-hydroxy-quinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X Inhibitor (or)present 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside X X color BlackTeal (or) Magenta

Example I

The microorganisms chosen to be identified, quantified anddifferentiated are E. coli, general coliforms, Aeromonas, Shigella orSalmonella.

With reference to Table IV, E. coli produces the enzyme Bgluc, and Bglucis not produced by any of the other microorganisms desired to bedetected. With reference to the right hand side of Table V, it can beseen that the test enzyme Bgluc has a corresponding substrate componentof β-D-glucuronide. Thus, a chromogenic or nonchromogenic componentwhich produces a distinct color upon cleavage of Bgluc should be chosenfrom the left hand side of Table V. 8-hydroxyquinoline is chosen for itspreferred substantially black color. The first identified substrate istherefore 8-hydroxyquinoline-β-D-glucuronide, the availability of whichis described above. A metallic salt such as ferric ammonium citrate isalso required and is added to the test medium so that, upon cleavage ofthe substrate by Bgluc, a substantially black water-insoluble complexforms in the medium. The substantially black precipitate consists of theferric ions and the aglycone released when the substrate is hydrolyzedby the glucuronidase from E. coli.

With firther reference to Table IV, Bgal, Bfuc and Bglu are common toAeromonas and general coliforms. However, as indicated in Table IV,Bgal, Bfuc and Bglu are not produced generally by Salmonella andShigella. Therefore, a substrate component corresponding to one of Bgal,Bfuc and Bglu can be selected form the right hand side of Table V. Bgaland the associated substrate component β-D-galactopyranoside are chosen.The 6-chloro-3-indolyl-chromogenic component produces a red-pink colorupon cleavage from its substrate in the presence of Bgal and is selectedas the chromogenic component. The second substrate is therefore6-chloro-3-indolyl-β-D-galactopyranoside.

Again referring to Table IV, Bman, Aara and Agal are common toSalmonella, Shigella and general coliforms. However, as indicated inTable IV, Bman, Aara and Agal are not produced by Aeromonas. Thus, oneof Bman, Aara and Agal can be chosen and its associated substratecomponent identified with reference to Table V. The test enzyme Agal andthe respective substrate component α-D-galactopyranoside are chosen.Next, a chromogenic component must be selected from Table V. As shown onthe left hand side of Table V, the chromogenic component5-bromo-4-chloro-3-indolyl produces a teal color upon cleavage from itsassociated substrate and is therefore selected. The third substrate istherefore 5-bromo4-chloro-3-indolyl-α-D-galactopyranoside.

General coliforms have a wide enzyme pattern which is responsive to boththe 6-chloro-3-indolyl-β-D-galactopyranoside substrate and the5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside substrate. Therefore,general coliforms will show as a fourth distinct color which is acombination of the colors produced by the two aforementioned substrates,respectively. In this case the fourth color will be violet-blue, whichis a combination of red-pink and teal.

Finally, as seen in Table IV, E. coli also exhibits a wide enzymepattern and responsive to all three of the substrates chosen in thisexample, viz., 8-hydroxyquinoline-β-D-glucuronide,6-chloro-3-indolyl-β-D-galactopyranoside, and5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside. Nonetheless, E. colicolonies present in the test medium will show as a substantially blackcolor because, as discussed above, the chromogenic substrates do notinterfere with the substantially black color. Advantageously, thissubstantially black color provides a superior means for distinguishingthe E. coli, as well as allows four separate microorganisms to bedetected, quantified, differentiated and identified in a single testmedium. See Table VI.

Example II

The selected microorganisms to be detected, quantified, differentiatedand identified are E. coli as a first color; general coliforms,Salmonella and Shigella as a second color; and Aeromonas as a thirdcolor.

With reference to Table IV, E. coli produces the enzyme Bgluc, and Bglucis not produced by any of the other microorganisms desired to bedetected. With reference to the right hand side of Table V, it can beseen that the test enzyme Bgluc has a corresponding substrate componentof β-D-glucuronide. Thus, a chromogenic or nonchromogenic componentwhich produces a distinct color upon cleavage of Bgluc should be chosenfrom the left hand side of Table V. 8-hydroxyquinoline is chosen for itspreferred substantially black color. The first identified substrate istherefore 8-hydroxyquinoline-β-D-glucuronide, the availability of whichis described above. A metallic salt such as ferric ammonium citrate isalso required and is added to the test medium so that, upon cleavage ofthe substrate by Bgluc, a substantially black water-insoluble complexforms in the medium. The substantially black precipitate consists of theferric ions and the aglycone released when the substrate is hydrolizedby the glucuronidase from E. coli.

With further reference to Table IV, Bgal, Bfuc and Bglu are common toAeromonas and general coliforms. However, as indicated in Table IV,Bgal, Bfuc and Bglu are not produced by Salmonella or Shigella. UsingTable V in the fashion described above,6-Chloro-3-indolyl-β-D-galactopyranoside is selected as the secondsubstrate, which will produce a red-pink color upon cleavage asindicated by the chromogenic component list of Table V.

As seen in Table IV, the enzyme Bman is common to Salmonella/Shigellabut not Aeromonas. From table V, the substrate component associated withBman is β-D-mannopyranoside. In this example, it is desired to alsoproduce the second distinct color (red-pink) with Salmonella/Shigella sothat, ultimately, Salmonella/Shigella colonies present in the testmedium will show as the same color as general coliforms present in thetest medium Thus, the chromogenic component is 6-Chloro-3-indolyl- andthe third substrate is therefore 6-Chloro-3-indolyl-β-D-mannopyranoside.

In this example, again using Table V, a fourth substrate is identifiedthat will be cleaved by one of the enzymes Bman, Aara, Agal common toSalmonella/Shigella to produce a third distinct color. Using table V inthe fashion described above, the fourth substrate selected is5-Bromo4-chloro-3-indolyl-α-D-galactopyranoside, which produces ateal-green color in the presence of Agal common to Salmonella/Shigella.

The resulting colors of colonies present in the test medium can bepredicted as follows. E. coli exhibits a wide enzyme pattern that ispositive for all four of the substrates chosen in this example,including the 8-hydroxy-glucuronide substrate which produces asubstantially black color upon cleavage in the presence of the ions ofthe ferric salt. E. coli colonies show as substantially black. Aeromonashas an enzyme pattern which reacts with only the6-Chloro-3-indolyl-β-D-galactopyranoside substrate chosen in thisexample and therefore colonies of Aeromonas show as red-pink.Salmonella/Shigella has an enzyme pattern which cleaves both the thirdand fourth substrates selected in this example and therefore colonies ofSalmonella/Shigella show as purple-blue (a combination of teal andred-pink). Finally, general coliforms are positive for each of thesecond, third and fourth substrates selected and colonies thereof showas purple-blue, indistinguishable from the Salmonella/Shigella colonies.As discussed above, different strains of all species of the variousgenera will not all produce the same amounts of the various enzymes, sothere may be slight variations in shades of purple-blue, for example.

Example IIIA

The selected microorganisms to be quantified and differentiated in thisexample are E. coli as a first color, general coliforms and Aeromonas asa second color, and Salmonella/Shigella as a third color.

With reference to Table IV, E. coli produces the enzyme Bgluc, and Bglucis not produced by any of the other microorganisms desired to bedetected. With reference to the right hand side of Table V, it can beseen that the test enzyme Bgluc has a corresponding substrate componentof β-D-glucuronide. Thus, a chromogenic or nonchromogenic componentwhich produces a distinct color upon cleavage of Bgluc should be chosenfrom the left hand side of Table V. 8-hydroxyquinoline is chosen for itspreferred substantially black color. The first identified substrate istherefore 8-hydroxyquinoline-β-D-glucuronide, the availability of whichis described above. A metallic salt such as ferric ammonium citrate isalso required and is added to the test medium so that, upon cleavage ofthe substrate by Bgluc, a substantially black water-insoluble complexforms in the medium. The substantially black precipitate consists of theferric ions and the aglycone released when the substrate is hydrolizedby the glucuronidase from E. coli.

Using tables IV and V in a fashion similar to that described above withreference to Examples I and II, 6-Chloro-3-indolyl-β-D-galactopyranosideis selected as a second substrate to combine with one of the enzymesBgal, Bfuc and Bglu common to coliforms and Aeromonas, but negative forSalmonella/Shigella to produce a second distinct color, in this casesubstantially red-pink.

Similarly, 6-Chloro-3-indolyl-α-D-galactopyranoside is selected as athird substrate to combine with Agal, which is common to coliforms andSalmonella/Shigella, but negative for Aeromonas. Upon reaction with theenzyme, this substrate will also produce the same distinct second color,namely red-pink.

5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside is selected as a fourthsubstrate to combine with the enzyme Bgal, which is common to coliformsand Aeromonas, but negative for Salmonella/Shigella. This fourthsubstrate produces a teal-green color upon reaction with Bgal.

The resulting colors of colonies present in the test medium can bepredicted as follows. E. coli exhibits a wide enzyme pattern and ispositive for all four of the substrates chosen in this example.Therefore, E. coli colonies will show as substantially black. Generalcoliform colonies have an enzyme pattern which is positive for thesecond, third and fourth substrates, so that general coliforms coloniesshow as purple-blue. Aeromonas colonies have an enzyme pattern which ispositive for the second and fourth substrates chosen so that Aeromonascolonies also show as purple-blue. Finally, the enzymes common toSalmonella/Shigella are only positive for the third of the foursubstrates, so that Salmonella/Shigella colonies show as red-pink.

Example IIIB

As a variation, the test medium of Example IIIA can be prepared suchthat colonies of Salmonella/Shigella will show as teal instead ofpink-red, all of the other colony colors being the same as Example IIIA.With reference to Table VI, such can be accomplished by replacing the6-chloro-3-indolyl-α-D-galactopyranoside of Example IIIA with5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside.

Example IIIC

A second, independent method for producing the same three colors asExample IIIA for the same four components can be achieved by addingnalidixic acid or other antibiotics or inhibitors of Aeromonas to thecomponents listed in Example 1. In so doing, the cefsulodin or nalidixicacid or other substance acts as an inhibitor for Aeromonas so Aeromonascolonies do not grow. If Aeromonas is eliminated, then the purple-bluecolonies are all true coliforms. If not eliminated, any Aeromonas willbe counted as part of the coliforms which some persons may prefer sinceAeromonas is an important indicator organism.

Example IV

In this example, the selected microorganisms to be detected, quantifiedand differentiated are E. coli, coliforms and Salmonella/Shigella as afirst distinct color and Aeromonas as a second distinct color. One testmedium which achieves this result is the test medium described inExample II, except that the first substrate and metallic salt areomitted. Thus, because the enzyme pattern of E. coli reacts with thesame substrates as the enzyme pattern for general coliforms, E. coli andgeneral coliforms will be the same color in this test medium.Specifically, E. coli, coliforms and Salmonella/Shigella colonies willshow as a purple-blue color, whereas Aeromonas colonies will show as asubstantially red-pink color.

Example V

The selected microorganisms to be detected, quantified anddifferentiated are E. coli, general coliforms and Aeromonas as a firstdistinct color, and Salmonella/Shigella as a second distinct color. Onetest medium which achieves this result is the test medium of Example 3with the first substrate and metallic salt being omitted. In this testmedium, E. coli, general coliforms and Aeromonas colonies will show as agenerally purple-blue color, whereas Salmonella and Shigella colonieswill show as a generally teal-green color or as a red-pink color.

Optionally, the 6-chloro-3-indolyl-α-D-galactoside can be replaced with5-bromo-4-chloro-3-indolyl-β-D-galactoside so that Salmonella coloniesshow as teal, rather than pink.

A third way to achieve the same result is with an antibiotic, preferablynalidixic acid, to inhibit the growth of Aeromonas colonies. IfAeromonas is eliminated, then the purple-blue colonies are all truecoliforms. If not eliminated, any Aeromonas will be counted as part ofthe coliforms which some persons may prefer since Aeromonas is animportant indicator organism.

Example VI

The selected microorganisms to be detected, quantified anddifferentiated are E. coli and coliforms as a first distinct color,Aeromonas as a second distinct color and Salmonella/Shigella as a thirddistinct color. A test medium which achieves this result is the testmedium of Example I with the first substrate and metallic salt beingomitted. In such a test medium, E. coli and general coliform colonieswill show as purple-blue, Aeromonas colonies will show as generallyred-pink, and Salmonella or Shigella colonies will show as generallyteal-green.

Example VII

The selected microorganisms to be detected, quantified anddifferentiated are E. coli as a first distinct color which issubstantially black; general coliforms as a second distinct color whichis substantially purple-blue; Aeromonas/Vibrio/Plesiomonas as a thirddistinct color which is substantially red-pink; and Salmonella orShigella as a fourth distinct color which is substantially teal-green.

With reference to Table IV, E. coli produces the enzyme Bgluc, and Bglucis not produced by any of the other microorganisms desired to bedetected. Therefore, a substrate which produces a distinct color uponcleavage of Bgluc should be chosen from Table V.8-hydroxyquinoline-β-D-glucuronide produces a substantially black colorin the presence of Bgluc and would be the preferred choice of substrate,as explained below. A metallic salt such as ferric ammonium citrate isalso added to form a substantially black water insoluble complexconsisting of the ferric ions and the aglycone released when thesubstrate is hydrolyzed by the glucuronidase from E. coli.

With further reference to Table IV, it can be seen that the enzyme Ngaland Naglu are common to the microorganisms Aeromonas, Plesiomonas, andVibrios. Therefore, a suitable substrate for testing all of thesemicroorganisms as a single distinct color is6-chloro-3-indolyl-N-acetyl-β-D-galactosaminide, which produces asubstantially red-pink color in the presence of these enzymes.

Again referring to Table IV, Bman, Aara and Agal are common toSalmonella/Shigella and general coliforms. However, as indicated inTable IV, Bman, Aara and Agal are not produced by Aeromonas. Therefore,a substrate can be selected from Table V which reacts with one of Bman,Aara and Agal to produce a third distinct color. As shown in Table V,5-bromo-4-chloro-3-indolyl-α-D-galactoside produces a teal-green colorin the presence of Agal and is therefore selected as a substrate.

In this test medium E. coli colonies will show as substantially black,general coliform colonies will show as substantially purple-blue,Aeromonas, Vibrio and Plesiomonas colonies will show as substantiallyred-pink, and Salmonella or Shigella colonies will show as substantiallyteal.

Example VIII

The selected microorganisms to be detected, quantified anddifferentiated are E. coli as a first distinct color; coliforms,Salmonella or Shigella as a second distinct color; and Aeromonas,Virbrio and Plesiomonas as a third distinct color. One test medium forachieving this result is the test medium of Example 2, except that thefourth substrate chosen is6-Chloro-3-indolyl-N-acetyl-α-D-galactosaminide, to which each of themicroorganisms Plesiomonas, Vibrios and Aeromonas are responsive so thateach of these colonies shows as a generally red-pink color.

Example IX

The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli and general coliforms as afirst distinct color which is purple-blue; Aeromonas, Plesiomonas, andVibrios as a second distinct color which is red-pink; and Salmonella orShigella as a third distinct color which is teal-green. This result canbe achieved with the test medium as described in Example 6 with theaddition of 6-Chloro-3-indolyl-N-acetyl-β-D-galactosaminide, to whicheach of the microorganisms Plesiomonas, Vibrio and Aeromonas isresponsive.

Example X

The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli and general coliforms as afirst color; Aeromonas, Vibrio and Plesiomonas as a second distinctcolor; and Salmonella or Shigella as a third distinct color. A suitabletest medium that achieves this result is the test medium disclosed inExample 7 except that the first substrate for detecting E. coli coloniesis omitted. In this example, E. coli and general coliform colonies showas generally purple-blue, Aeromonas, Vibrio and Plesiomonas show asgenerally red-pink, and Salmonella or Shigella show as generallyteal-green. The addition of 6-Chloro-3-indolyl-β-D-galactopyranoside isnecessary to yield the purple-blue color for E. coli colonies.

Example XI

The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli as a substantially blackcolor and general coliforms as a red-pink color.

With reference to Table IV, E. coli produces the enzyme Bgluc, and Bglucis not produced by any of the other microorganisms desired to bedetected. Therefore, a substrate which produces a distinct color uponcleavage of Bgluc should be chosen from Table V.8-hydroxyquinoline-β-D-glucuronide produces a dark color in the presenceof Bgluc and would be the preferred choice of substrate. A metallic saltsuch as ferric ammonium citrate is also added to form a black waterinsoluble complex consisting of ferric ions and the aglycone releasedwhen the substrate is hydrolized by glucuronidase from E. coli.

With further reference to Table IV, Bgal, Bfuc and Bglu are common toAeromonas and general coliforms. However, as indicated in Table IV,Bgal, Bfuc and Bglu are not generally produced by Salmonella orShigella. Therefore, a substrate can be selected from Table V whichreacts with one of Bgal, Bfuc and Bglu to produce a second distinctcolor. 6-chloro-3-indolyl-β-D-galactopyranoside produces a pink color inthe presence of Bgal and is selected as the second substrate.

Optionally, the 6-chloro-3-indolyl-β-D-galactopyranoside can be replacedwith 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside so that Aeromonasand general coliform colonies show as teal instead of pink.

As noted, the second substrate selected will result in colonies ofAeromonas also showing as a generally red-pink color. To avoid growth ofAeromonas colonies, an inhibitor, preferably nalidixic acid, is added.Thus, colonies of E. coli will show as substantially black, whereascolonies of general coliforms will show as a red-pink color.

Example XII

The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli, general coliforms andAeromonas spp. as a substantially black color and Salmonella or Shigellaspp. as a second distinct color. The first substrate selected is8-hydroxyquinoline-β-D-galactoside, which results in colonies of E.coli, general coliforms and Aeromonas showing as substantially black.The second substrate chosen can be either5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside or6-chloro-3-indolyl-α-D-galactopyranoside. If the former of these twosubstrates is chosen, colonies of Salmonella or Shigella will show as ateal color, whereas if the latter of the two aforementioned substratesis chosen, colonies of Salmonella or Shigella will show as a red-pinkcolor.

Optionally, in this example, Aeromonas may be eliminated by adding aninhibitor, preferably nalidixic acid, as discussed in detail above.

Example XIII

The selected microorganisms to be detected, quantified anddifferentiated in this example are the same as in Example 1, except thatthis example illustrates a correction for false positives. That is, itis possible that certain unusual Enterobacter and Klebsiella spp. willshow as black colonies along with E. coli in the test medium disclosedin Example 1. Thus, the count of E. coli could be inaccurately high.

In this example, 4-methyl-umbrelliferyl-β-D-xylopyranoside is added tothe test medium described in Example 1. In so doing, Enterobacter andKlebsiella spp. showing as black colonies will also fluoresce, therebyallowing reduction in the false positive count of E. coli. This exampleillustrates the flexibility of embodiments incorporating the presentinvention. The fluoroescent component does not interfere with thesubstantially black color so that the black colonies are easilydistinguished with the naked eye. Yet, under ultraviolet light, falsepositives can be detected and substantially reduced by examining theblack colonies for fluorescence.

Example XV

The selected microorganisms to be detected, quantified, differentiatedand identified are E. coli as a substantially black color and Salmonellaspp. as pink-red. General coliforms are colorless in this example.

With reference to Example I, E.Coli is responsive to8-hydroxy-quinoline-β-D-glucuronide. General coliforms, Salmonella,Shigella and Aeromonas are not responsive to8-hydroxy-quinoline-β-D-glucuronide. Thus, the first substrate chosen is8-hydroxy-quinoline-β-D-glucuronide.

With reference to table IV, esterase enzyme is positive for Salmonellaspp., but not any of the other preferred microorganisms to be detected.With reference to table V, the substrate 6-chloro-3-indolyl-caprylatecan be identified, and will produce a pink-red color upon cleavage, andis therefore chosen as the second substrate.

In this test medium, colonies of E. coli will show as substantiallyblack and colonies of Salmonella or Shigella will show as pink-red.

Example XV

The selected microorganisms to be detected, quantified, differentiatedand identified are E. coli as a substantially black color, Salmonellaspp. as dark blue-purple, and general coliforms as red-pink.

With reference to Example I, E. coli is responsive to8-hydroxy-quinoline-β-D-glucuronide. General coliforms, Salmonella,Shigella and Aeromonas are not responsive to8-hydroxy-quinoline-β-D-glucuronide. Thus, the first substrate chosen is8-hydroxy-quinoline-β-D-glucuronide.

With reference to tables IV and V, 5-bromo-4-chloro-3-indolyl-caprylatecan be identified as the second substrate to which Salmonella orShigella will be responsive. With further reference to Table V,5-bromo-4-chloro-3-indolyl-caprylate forms a teal color upon cleavage.

6-chloro-3-indolyl-α-D-galactopyranoside, which produces a pink-redcolor upon cleavage, is chosen as the third substrate to which E.coligeneral coliforms and Salmonella or Shigella are responsive.

In this example, E. coli colonies show as substantially black, generalcoliform colonies show as red-pink, and Salmonella/Shigella show asblue-violet (=red-pink+teal).

Example XVI

The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli as a substantially blackcolor and general coliforms as a second distinct color.

With reference to Table IV, E. coli produces the enzyme Bgluc, and Bglucis not produced by any of the other microorganisms desired to bedetected. Therefore, a substrate which produces a distinct color uponcleavage of Bgluc should be chosen from Table V.8-hydroxyquinoline-β-D-glucuronide produces a dark color in the presenceof Bgluc and would be the preferred choice of substrate. A metallic saltsuch as ferric ammonium citrate is also added to form a black waterinsoluble complex consisting of ferric ions and the aglycone releasedwhen the substrate is hydrolized by glucuronidase from E. coli.

With further reference to Table IV, Bgal, Bfuc and Bglu are common toAeromonas and general coliforms. However, as indicated in Table IV,Bgal, Bfuc and Bglu are not generally produced by Salmonella/Shigella.Therefore, a substrate can be selected from Table V which reacts withone of Bgal, Bfuc and Bglu to produce a second distinct color.5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside can be chosen as thesecond substrate, in which event colonies of E. coli will appear assubstantially black and general colifom colonies will appear as teal.Optionally, 5-bromo-6-chloro-3-indolyl-galactopyranoside can be chosenas the second substrate, in which event colonies of E. coli will appearas substantially black and general colifom colonies will appear magenta.To avoid growth of Aeromonas colonies, an inhibitor, preferablynalidixic acid, is added. Thus, colonies of E. coli will show assubstantially black, whereas colonies of general coliforms will show asa magenta color.

Although several broad examples which incorporate the present inventionhave been described above, it is to be understood that the presentinvention is not to be limited by the examples disclosed herein. Indeed,the disclosure and examples above teach one of ordinary skill avirtually limitless number of test media which would be within the scopeof the claims appended hereto.

Further, while this invention has been described as having a preferreddesign, the present invention can be further modified within the spiritand scope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the invention using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims.

What is claimed is:
 1. A method for detecting, quantifying, anddifferentiating colonies of Aeromonas from selected other biologicalentities in a test sample, said method comprising the following steps:providing a base medium including ions of a salt, a β-D-galactosidesubstrate that forms a first component of a first color in the presenceof a first enzyme, and an α-D-galactoside substrate that forms a secondcomponent of a second color distinguishable from said first color in thepresence of a second enzyme; inoculating the test medium with a testsample; incubating the test medium; and examining the test mediumwhereby aggregations of colonies of Aeromonas are indicated by saidfirst color, and aggregations of colonies of Salmonella are indicated bysaid second color, and whereby colonies of general coliforms areindicated by a third color, said third color being a combination of saidfirst and second colors.
 2. The method as set forth in claim 1, wherebycolonies of E. coli are also indicated by said third color.
 3. Themethod as set forth in claim 2, wherein said β-D-galactoside substrate dsaid α-D-galactoside substrates are chromogenic substrates.